Anti-inflammatory and anti-neuropathic effects of a novel quinic acid derivative from Acanthus syriacus.

Objective
Acanthus syriacus (AS) is one of the valuable herbal plants with immunomodulatory potentials. The aim of this study is to assemble a phytochemical investigation of A. syriacus exploring its anti-inflammatory and antinociceptive properties, identification of its most active compound(s) and elucidating their structure and determining their mechanisms of action.


Materials and Methods
Bio-guided fractionation and isolation-schemes were used utilizing RP-HPLC, CC, 1H- and 13C-NMR, and biological-models were used to evaluate their effects against inflammation and neuropathic-pain (NP).


Results
The outcomes showed that the most active fraction (FKCA) of AS was identified. Two of the three components of FKCA were identified by chromatographic-methods, while the third compound was isolated, its structure was elucidated and its was named Kromeic acid (KRA); FKCA contained Ferulic acid (27.5%), kromeic acid (48.1%), and chlorogenic acid (24.4%). AS, FKCA and KRA showed significant (p˂0.05) anti-inflammatory and antinociceptive potentials in the management of allodynia and thermal-hyperalgesia in NP. AS and FCKA showed comparatively equipotent antinociceptive-effects. FKCA showed higher antinociceptive effects than KRA suggesting additive-effects among FKCA components. The anti-inflammatory, insulin secretagogue, oxidative-stress reducing, and protective effects against NO-induced neuronal-toxicity might be amongst the possible mechanisms of tested compounds to alleviate NP.


Conclusion
Here, we report the isolation and structure elucidation of a novel quinic-acid derivative, KRA. A. syriacus, FKCA, and KRA might be used as a novel complementary approach to ameliorate a variety of painful-syndromes.


Introduction
The Acanthus genus belonging to the Acanthaceae family which is a large plant family consisting of 250 genera and 2700 species distributed widely across the Mediterranean and tropical regions of the world (Capanlar et al., 2010). The majority of the Acanthus species have been used in Asian traditional medicine for amelioration of various ailments. Various Acanthus species have shown anti-hepatotoxic, antioxidant, antimicrobial, antitumor, antiviral, anti-inflammatory, analgesic, and anti-fertility activities (Asongalem et al., 2004;Babu et al., 2001Babu et al., ,2002Bravo et al., 2004;Capanlar et al., 2010). The major constituents of various Acanthus species were determined to be alkaloids, flavonoids, glycosides, saponins, and lignans (Bravo et al., 2004;Capanlar et al., 2010). In Lebanon, Acanthus syriacus is one of the endemic species that has been used as a folk medicine for its immunomodulatory properties and against various neurological disorders (Baydoun et al., 2015).
The carrageenan in-vivo experiments are widely accepted-models for assessment of the anti-inflammatory effects of compounds and are principally used for the evaluation of the acute-anti-inflammatory potentials of natural or synthetic compounds (Willoughby and DiRosa, 1972).
Neuropathic pain (NP) is amongst the most difficult pain types to treat after being provoked as a complication of many ailments including diabetes (Ziegler, 2008). High blood glucose level was shown to provoke allodynia and hyperalgesia in response to chemical or thermal nociceptive provocation (Pabbidi et al., 2008). The NP pathogenesis is multifactorial and the involved mechanisms are not fully understood (Taliyan and Sharma, 2012). Hyperglycemia also causes reactiveoxygen species (ROS) over-production and innate antioxidant defenses aggravation, increasing the oxidative stress which is an NP fundamental mechanism (Ozkul et al., 2010). Hyperglycemia provoked oxidative stress and advanced glycation end-product generation leading to stimulation of proinflammatory cytokines (CKs) (Cameron and Cotter, 2008). Pro-inflammatory CKs provoke nitric oxide (NO) synthase expression and elevate NO production (Yu et al., 2009;Taliyan and Sharma, 2012). Moreover, NO is involved in allodynia and hyperalgesia causing the NP (Joharchi and Jorjani, 2007;Taliyan and Sharma, 2012).
Currently, different classes of nonsteroidal anti-inflammatory drugs, opioids, antidepressants, and anticonvulsants are used for amelioration of NP; nevertheless, the limited pain relief is achieved due to their partial-efficiency and recorded toxicities (Ziegler, 2010). Thus, there is an increasing need to discover more efficient and safer drugs for the management of NP. Alternative medicines have gained a reputation in the management of NP, and many indigenous medicinal plants were found to be effective in ameliorating NP (Comelli et al., 2009;Raafat et al., 2017b;Raafat and Hdaib, 2017;Taliyan and Sharma, 2012). Moreover, there is an increasing necessity for deeper investigation of these complementary medicines to understand their chemical compositions and identify their potential compounds and their mechanisms of actions to be used for NP amelioration.
A. syriacus is a promising herbal remedy. However, to date, there are no deep phytochemical or biological reports about A. syriacus.
Therefore, the aim of the present study is to assemble a phytochemical investigation of A. syriacus exploring its antiinflammatory and antinociceptive properties, identification of its most active compound(s), elucidating active compounds structure and discovering their possible mechanisms of action.

Materials and Methods
Standards and solvents were commercially obtained from Merck-Sigma-Aldrich (Germany).

Plant material
The aerial parts of A. syriacus were collected from Yahchouch, Kesrwan, Mount Lebanon (N 34° 04` 09`` E 35° 44` 20``, Lat: 34.0692911 Lng: 35.7387591), during the flowering stage in the middle of May 2016. The specimen was authenticated with a reference sample and a specimen was deposited in the faculty herbarium with the voucher .

Extraction
The aerial parts of A. syriacus were dried in shade and size-reduced by G. Ming Mill (China) to form a powder. The dried powder was defatted by hexane and then, sonicated twice using 80% ethanol for 6hr at room temperature. Then, the extract was dried under vacuum at 40°C by Buchi rotary-evaporator (Germany) and then lyophilized utilizing Edwards freeze drier (Germany). The extract was kept frozen at-40°C until further utilization.

A. syriacus Bio-guided fractionation and isolation
The extract was then fractionated utilizing silica gel column-chromatography (75×15 cm). The column was developed by a gradient mobile phase: one bed volume (BV) of hexane/ethyl acetate (50:50, v/v), then one BV EtAc, then one-BV of EtAc/water/formic acid (46:46:8, v/v), then 2 BV of EtAc, formic acid, water and hexane (70:7.5:7.5:15,v/v/v/v),then 1 BV ethanol/water (50:50,v/v), and finally one BV 100% water. Fractions were collected every 2 min. and similar fractions were combined and concentrated under reduced pressure. In order to identify the most active compound (s), each fraction was evaluated in the same-way as the A. syriacus extract for its antinociceptive properties. Most fractions were identified by steeping method utilizing the RP-HPLC system and utilizing reference standards. Peak nine was (Figure 1) identified utilizing 1 H and 13 C NMR analysis.

Sample preparation for 1 H and 13 C NMR investigation
The NMR experiments were done using a Bruker 300 MHz spectrometer (Germany) equipped with an auto-sampler. NMR samples were prepared by dissolving the isolated compound(s) in deuterated methanol (MeOH-d4, Sigma-Aldrich). NMR spectra 13 C-HSQC (Hetero-nuclear single quantum coherence), 13 C-HMBC (Hetero-nuclear Multiple Bond coherence), ROESY NMR (Rotating-frame Overhauser Effect Spectroscopy) and COSY NMR (Correlation Spectroscopy) were obtained at 300 MHz, at 25°C.

Animals
Male albino mice (22-30g) were accommodated for one week prior to the in vivo experiment. The animals had freeaccess to water and standard feeding pellets (except otherwise stated), and were kept under 12hr/12hr dark/light cycles. All experiments were done according to animal-care rules and regulations, and approved by BAU Institutional Review Board (2019A-0056-P-R-0297).

Acute carrageenan-provoked inflammatory-pain
In order to assess the acute carrageenaninduced inflammatory-pain, 100μL of 1% carrageenan-solution was intraplantarly injected into the mice left hind-paw (n=7/group).

Diabetes induction
Diabetes was induced in mice by injecting 180mg/kg alloxan every other day for three days. The blood glucose level (BGL) was checked by pricking the animal tail and utilizing Accu-chek glucometers (Germany) (Jamalan et al., 2015;Khaneshi et al., 2013). The blood glucose levels (BGL) were measured acutely at hour 6 and subchronically for 8 days, and for 8 weeks. Sigma glucometers (Germany) were utilized to monitor BGL. The glycatedhemoglobin (HbA1c) level was monitored utilizing Analyticon HbA1c kits (Germany) for 8 weeks. Animals having BGL≥200 mg/dL and HbA1c > 8 were considered diabetic.
-Vehicle control (VEH or DIA+VEH): Group of alloxan-induced vehicle-treated diabetic control mice.

Nociceptive responses assessment
Eight weeks after diabetes induction, animals were evaluated for diabeticneuropathy success-rate (DNSR, significant losing sensory response to thermal-nociception below 10 sec (Sullivan et al., 2007). The DNSR was about 88% and the test compounds antinociceptive potentials were evaluated every other week for eight weeks.

Thermal-nociceptive latency evaluation
Animals with DNSR were involved in the thermal-hyperalgesia tail-flick and hotplate latency experimentations (Micov et al., 2015). In brief, the animals were tested utilizing a hot-plate analgesia-meter (Ugo-Basile-Italy), or a tail-flick apparatus (Hugo-Sachs-Elektronik-Germany). The thermal-intensity was tuned to provide a baseline latency-time for the hot-plate test of 4-5 sec, and 1.5-2.5 sec for the tail-flick test for normal non-diabetic mice (NORM). A 10-sec cut-off time was adopted in thermal-nociceptive latency experiments to avoid tissue-damage.

Mechanical-nociceptive latencies evaluation
The tactile-allodynia, in mice with DNSR, was evaluated by monitoring the paw-withdrawal-thresholds (PWT) using Von-Frey-filaments (OptiHair) (Ohsawa et al., 2011). In brief, mice were separately put on a mesh floor in a bottom-up plastic cage. The force on the plantar-surface of the animal left hind-paw was gradually increased till it withdrew the paw. Here, 32 g cut-off force was adopted in the mechanical-nociceptive experiments for animal safety.

Nitric oxide (NO) levels measurement
NO level was determined by utilizing the Griess-reagent technique (Green et al., 1982). Both urinary (µM/L) and tissue (µM/mg protein) nitrite were measured.

Statistical analysis
Outcomes (mean ± SEM) were statistically assessed by one way ANOVA followed up by the Student-Newman-Keuls analysis utilizing OriginPro® statistics-software. Ap-value˂0.05 was regarded as statistically-significant.

A. syriacus bio-guided isolation, RP-HPLC, 1 H and 13 C NMR structure elucidation and identification of the most active compounds
The most active fraction having antinociceptive potentials was isolated utilizing column-chromatography separation method. The RP-HPLC analysis, similar to the whole extract, was done for the most active fraction. Three compounds were recognized in this active fraction. Two compounds of the most active fraction were identified as ferulic acid and chlorogenic acid by steeping method utilizing reference standards. The third compound was isolated from the mixture by subjecting it to second silica-gel column-chromatography and was developed using dichloromethane and mixtures of dichloromethane and methanol with increasing polarities. Similar fractions were combined and concentrated. The third compound was further purified by semipreparative HPLC. Compound 3 was obtained as an amorphous yellowish solid and identified by m/z: 340.1200 using Nano-ESI-MS in the positive mode, elemental analysis: C, 56.5%; H; 5.9%; O, 37.6%, and chemical formula C16H20O8. The 1 H Spectral data ( The methylene group protons appeared as two duplets at δ 3.81. The main protonproton neighboring interactions of the aromatic ring were detected between H-29 and H-30 in the COSY spectrum. The 13 C NMR chemical shifts (Table 2) at δ114.8, δ 116.1, δ 123.0, δ 130.7, δ 132.3, δ 146.5, and δ 146.5 were assigned to the aromatic carbons. Comparison of the 13 C NMR with HMQC and DEPT spectra revealed C13 methylene group. The olefinic group carbon appeared at δ123.1 and δ132.3. The quinic acid carbons appeared at δ38.1, δ38.3, δ70.3, δ71.8, δ73.5, andδ74.5, with the carboxylic acid carbon observed at δ 180.4. TheC13 signal of the methylene group at δ66.6 exhibited a clear correlation with the olefinic proton at δ 123.1 in the HMQC spectrum. The HMBC spectrum of the compound expounded a signal between the C2of the quinic acid and the H35of the methylene group (Table 2 and Figure 2). Therefore, the quinic acid derivative (compound 3) was named kromeic acid (KRA), and the most active fraction in A. syriacus (AS) was named FKCA due to its content of ferulic acid (27.5%), kromeic acid (48.1%), and chlorogenic acid (24.4%).

Thermal-pain responses
After 8 weeks of oral administration of different treatments, as compared to VEH, the orally-administrated AS 50, 100 and 150mg/kg provoked a significant rise in the thermal-stimuli reaction-time by 0.88, 1.07 and 1.00 folds for HPL, and by 1.44, 1.93 and 2.21 folds for TFL, respectively (Figures8A and 9A). Also, FKCA administration at doses of 5, 10 and 20mg/kg, raised HPL by 0.96, 1.01 and 1.07 folds, and elevated TFL by 1. 30,1.79,and 2.16 folds,respectively (Figures 8B and 9B). Moreover, KRA oral administration at doses of 5, 10 and 20mg/kg raised HPL by 0.53, 0.65, and 0.80 folds, and raised TFL by 1.10, 1.47 and 1.86 folds, respectively (Figures 8C and 9C). These outcomes were evaluated against tramadol 10mg/kg (TRA), a positive control, which increased HPL by 0.94 folds, and TFL by 1.69 folds (Figures  8 and 9). The efficiency of AS, FKCA, and KRA on amelioration of thermal hyperalgesia, proposes their antinociceptive properties against hyperalgesic-pain.

AS, FKCA and KRA potentials against urinary and tissue nitrite level
One of the possible anti-neuropathic pain mechanisms of action of a given compound is mediated through it potential to reduce nitrite level (Taliyan and Sharma, 2012). Thus, nitrite levels in the urine and heart left ventricle tissues were monitored pre-(control group) and 8-weeks posttreatment (test group), in order to explore the test compounds' antinociceptive mechanism.

Group
Dose (

Discussion
The phytochemical investigation, the bio-guided fractionation, and isolation schemes conducted in this study utilizing various chromatographic, NMR, and biological inflammatory and NP models, showed that A. syriacus most active fraction was the isolated FKCA fraction. Chromatographic methods identified ferulic and chlorogenic acids as two of the three components of FKCA by. The third compound was a quinic acid derivative elucidated by various analytical methods especially, 1 H and 13 C NMR method. The quinic acid derivative (compound 3) was named kromeic acid (KRA), and the most active fraction in A. syriacus (AS) was FKCA due to its content of Ferulic acid (27.5%), kromeic acid (48.1%), and chlorogenic acid (24.4%).
Moreover, the significant efficiency of the tested compounds with respect to raising SIL, implies that the insulinsecretagogue activity is amongst diabetes controlling mechanisms of AS, FKCA, and KRA. These results are similar to previous reports indicating Prunus cerasus and Anemone coronaria secretagogue potential as one of their anti-diabetic mechanisms of action (Raafat and El-Lakany, 2018;Saleh et al., 2017).
The antioxidant potentials of AS, FKCA, and KRA on elevating CAT and GSH, and reducing LPO levels, propose their potentials in ameliorating painfulneuropathy. Earlier studies on compounds with similar antioxidant potentials, also found antinociceptive properties (Muthuraman et al., 2008;Nishiyama and Ogawa, 2005).
AS, FKCA, and KRA ameliorating potentials on mechanical-nociceptive propose their antinociceptive activities against allodynic-pain. These results are aligned with earlier studies that suggested a neuroprotective effect for other naturalcompounds, like Curcuma sesquiterpenes and Gingko biloba polyphenolics, against neurotoxicity (Hibatallah et al., 1999;Raafat and Omar, 2016;Shi et al., 2010). Also, AS, FKCA, and KRA protective effects against NO-induced neuronal toxicity might be one of the test compounds' possible antinociceptive mechanisms as described before for G. biloba extract and other natural compounds (Green et al., 1982;Taliyan and Sharma, 2012).
AS, FCKA, and KRA showed significant anti-inflammatory activities. Compared to TRA, highest doses of AS, FKCA, and KRA showed higher antinociceptive potentials in controlling allodynic and thermal-hyperalgesic NP. AS and FCKA showed relatively equipotent antinociceptive effects with a predominant ameliorating effect of FCKA at its highest dose (20mg/kg). FKCA had more marked antinociceptive effects than KRA suggesting additive effects of FKCA components strengthening its antineuropathic potentials. The insulinsecretagogue, anti-inflammatory, antioxidative-stress, and protective effects against NO-induced neuronal toxicity potentials may be amongst AS, FKCA and KRA mechanisms through which they alleviated neuropathic-pain.
After conducting clinical-trials, A. syriacus, FKCA, and KRA might be used as a novel complementary approach for alleviation of a variety of painfulsyndromes.